1. Field of the Invention
The present invention relates to the detection, identification and monitoring of submicron size particles. More particularly, the invention pertains to apparatus and methods for the sampling, measuring, characterizing, automated detection, identification, and monitoring of submicron size particles. Preferably, the present invention provides for the sampling, detection and identification of submicron size particles having a size range of from about 5 to about 1000 nanometers. Such particles include viruses and virus-like agents (such as, for example, prions, viral subunits, viral cores of delipidated viruses, plant viruses, etc.), and other biological materials such as nanometer size portions of bacteria.
2. Fields of Use of the Invention
Detection and identification of viruses without limiting the detection and identification to a particular family, genus and species and searching for viruses pathogenic to humans in a single environment can be difficult.
As set forth in U.S. Pat. Nos. 6,051,189, 6,485,686, 6,491,872, and 7,250,138, assigned to the U.S. Government and herein incorporated by reference, viruses may also be extracted from an environment and concentrated to an extent that permits detection and monitoring of viruses, without culturing procedures. Generally, in the detection of small amounts of viruses in environmental or biological liquids, it may be necessary to both enrich the concentration of viruses many orders of magnitude (i.e., greatly reduce the volume of liquid containing the viruses) and accomplish removal of non-viral impurities. In the presence of non-viral impurities, even the most sensitive detection methods generally require virus concentrations on the order of 10 femtomoles/microliter or more in the sampled liquid to reliably detect the viruses.
Sampling for airborne viruses is generally accomplished by collecting airborne particles in liquid, using a process such as air scrubbing, or eluting from filter paper collectors into a liquid medium. Collection and subsequent separation and detection methods are affected by the adsorption of viruses into solids in aerosols and liquids.
In contrast, when sampling liquids for viruses, in many cases no special equipment or processes may be necessary in order to collect a sample; for example, in sampling blood and other body fluids for viruses, only a standard clinical hypodermic needle may be needed. For sampling of bodies of water or other conveniently accessible liquids, sample collection may not be an issue at all, and in such cases the term “collector” is often applied to what is, in reality, a virus extraction step (such as collection on a filter).
Bacteria are different types of microorganisms than viruses. Viruses are a magnitude smaller in size than bacteria. Bacteria are classified in their own scheme. They have cell walls or are organized into cellular components and generally are considered to be among the self-sustaining organisms. Viruses require a living cell to invade in their life cycle. The technology and processes disclosed in U.S. Pat. Nos. 6,051,189, 6,485,686, 6,491,872, and 7,250,138 capitalize on the size and physical properties of the viruses to separate, count and characterize them. There is sufficient information from this characterization to identify them and perform investigative studies.
Bacteria are generally 0.5-1 microns wide and 2-3 microns long, and generally outside the physical ability of the apparatus disclosed in the referenced U.S. Pat. Nos. 6,051,189, 6,485,686, and 6,491,872. Bacteria have, however, interesting features that are in the proper size range for the apparatus to characterize. For example, gram-negative bacteria, named because of their inability to retain crystal violet-iodine complex stain, have rigid surface appendages called “pili.” These “hair-like” structures are around 7 nm in diameter and vary in length, up to 25 nm for the longer flagellae, which are other nanometer-sized structures that can be attached to the surface of bacteria. The pili are composed of structural protein sub-units called “pilins.” Some structures have only one structural protein unit, other pili are more complex and have several. These pili consist of a precise helical arrangement of one or more types of protein and as indicated may have different lengths for different bacteria. Choudhury, et. al (1999): Science 7 Aug. 99 285:1061 and David Eisenberg: How chaperones protect virgin proteins (Science 13 Aug. 99 285:1021), discuss crystal complexes associated with pilin subunits. Cell lysis breaks the cell into components. Lysis can be achieved by changes in pH, temperature, sonic treatment or by chemical means.
Processes for the separation and collection of pili from bacteria are generally set forth in U.S. Pat. Nos. 4,443,431 (Buchanan et. al.), 4,472,302 (Karkhanis), 4,702,911 (McMichael), 4,740,585 (Schmidt et. al.), 4,971,794 (Linggood et. al.), 5,612,036 (Hodges et. al.), 5,750,116 (Brinton), 5,968,769 (Green et. al.), and 6,291,649 (Lindberg et. al.), the teachings of which are fully incorporated herein by reference. Generally, heat, chemical activity and physical means can be used to release the pili from the bacteria. The pili can then be separated from the bacteria cells and counted as nanometer sized particles. Further, it is expected that different pili proteins for different bacteria species can be expected to be separated and identified. Identification of pili as nanometer sized particles and rapid identification and classification of the bacteria according to the nanometer sized characteristics of the pili is desired.